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The organization of proteins in space and time is essential to their function. To accurately quantify subcellular protein characteristics in a population of cells with regard for the stochasticity of events in a natural context, there is a fastgrowing need for imagebased cytometry. Simultaneously, the massive amount of data that is generated by imagecytometric analyses, calls for tools that enable pattern recognition and automated classification. In this article, we present a general approach for multivariate phenotypic profiling of individual cell nuclei and quantification of subnuclear spots using automated fluorescence mosaic microscopy, optimized image processing tools, and supervised classification. We demonstrate the efficiency of our analysis by determination of differential DNA damage repair patterns in response to genotoxic stress and radiation, and we show the potential of data mining in pinpointing specific phenotypes after transient transfection. The presented approach allowed for systematic analysis of subnuclear features in large image data sets and accurate classification of phenotypes at the level of the single cell. Consequently, this type of nuclear fingerprinting shows potential for highthroughput applications, such as functional protein assays or drug compound screening. © 2009 International Society for Advancement of Cytometry

Polymers adsorbing from a dilute solution onto the solvent−vapor interface generate a nonzero surface pressure. When the same polymers are end-grafted onto a surface such that a so-called polymer brush is formed, one will find that the solvent wets this compound interface partially. The partial wetting and the finite surface pressure are intimately linked properties of the polymer−solvent−vapor combination. It is shown that the spreading parameter in the wetting problem is proportional to the surface pressure in the adsorption case. Complete wetting is only possible when this surface pressure is nonpositive. The wetting characteristics are hardly influenced by the grafting density and chain length characterizing the brush. We argue that the grafted polymer chains can bridge to the solvent−vapor interface, thereby preventing the wetting film to become macroscopically thick. We present experimental data underpinning our self-consistent field analysis. Indeed, finite contact angles should be expected in various systems in which bridging attraction contributes to the disjoining pressure in wetting films.

The archaeal transcriptional initiation machinery closely resembles core elements of the eukaryal polymerase II system. However, apart from the established basal archaeal transcription system, little is known about the modulation of gene expression in archaea. At present, no obvious eukaryal-like transcriptional regulators have been identified in archaea. Instead, we have previously isolated an archaeal gene, the Pyrococcus furiosus lrpA, that potentially encodes a bacterial-like transcriptional regulator. In the present study, we have for the first time addressed the actual involvement of an archaeal Lrp homologue in transcription modulation. For that purpose, we have produced LrpA in Escherichia coli. In a cell-free P. furiosus transcription system we used wild-type and mutated lrpA promoter fragments to demonstrate that the purified LrpA negatively regulates its own transcription. In addition, gel retardation analyses revealed a single protein-DNA complex, in which LrpA appeared to be present in (at least) a tetrameric conformation. The location of the LrpA binding site was further identified by DNaseI and hydroxyl radical footprinting, indicating that LrpA binds to a 46-base pair sequence that overlaps the transcriptional start site of its own promoter. The molecular basis of the transcription inhibition by LrpA is discussed.

In the Lower Palaeozoic rocks of the Brabant Massif (Belgium), a recently discovered polysulphide mineralisation is intimately related to a high strain zone. Data from drillings, completed with outcrop data allow a detailed investigation of mineralisation, alteration and fluid characteristics of this high strain zone, currently interpreted as a low-angle reverse shear zone and attributed to the main Early to early Middle Devonian Acadian deformation event. Ore mineralisation occurred synkinematically and was closely associated with the shear zone. Low saline H2O–CO2(–CH4)–NaCl fluids with temperatures >260°C were involved in the hydrothermal circulation, which caused alteration of the host rock and extensive sericitisation in the shear zone. Isotope data and the general setting indicate a metamorphic-driven system.

Pyrococcus furiosus uses a modified Embden-Meyerhof pathway involving two ADP-dependent kinases. Using the N-terminal amino acid sequence of the previously purified ADP-dependent glucokinase, the corresponding gene as well as a related open reading frame were detected in the genome of P. furiosus. Both genes were successfully cloned and expressed in Escherichia coli, yielding highly thermoactive ADP-dependent glucokinase and phosphofructokinase. The deduced amino acid sequences of both kinases were 21.1% identical but did not reveal significant homology with those of other known sugar kinases. The ADP-dependent phosphofructokinase was purified and characterized. The oxygen-stable protein had a native molecular mass of approximately 180 kDa and was composed of four identical 52-kDa subunits. It had a specific activity of 88 units/mg at 50 degrees C and a pH optimum of 6.5. As phosphoryl group donor, ADP could be replaced by GDP, ATP, and GTP to a limited extent. The K(m) values for fructose 6-phosphate and ADP were 2.3 and 0.11 mM, respectively. The phosphofructokinase did not catalyze the reverse reaction, nor was it regulated by any of the known allosteric modulators of ATP-dependent phosphofructokinases. ATP and AMP were identified as competitive inhibitors of the phosphofructokinase, raising the K(m) for ADP to 0.34 and 0.41 mM, respectively.

ortho-Chlorophenol reductive dehalogenase of the halorespiring Gram-positive Desulfitobacterium dehalogenans was purified 90-fold to apparent homogeneity. The purified dehalogenase catalyzed the reductive removal of a halogen atom from the ortho position of 3-chloro-4-hydroxyphenylacetate, 2-chlorophenol, 2,3-dichlorophenol, 2,4-dichlorophenol, 2,6-dichlorophenol, pentachlorophenol, and 2-bromo-4-chlorophenol with reduced methyl viologen as electron donor. The dechlorination of 3-chloro-4-hydroxyphenylacetate was catalyzed by the enzyme at a Vmax of 28 units/mg protein and a Km of 20 microM. The pH and temperature optimum were 8.2 and 52 degrees C, respectively. EPR analysis indicated one [4Fe-4S] cluster (midpoint redox potential (Em) = -440 mV), one [3Fe-4S] cluster (Em = +70 mV), and one cobalamin per 48-kDa monomer. The Co(I)/Co(II) transition had an Em of -370 mV. Via a reversed genetic approach based on the N-terminal sequence, the corresponding gene was isolated from a D. dehalogenans genomic library, cloned, and sequenced. This revealed the presence of two closely linked genes: (i) cprA, encoding the o-chlorophenol reductive dehalogenase, which contains a twin-arginine type signal sequence that is processed in the purified enzyme; (ii) cprB, coding for an integral membrane protein that could act as a membrane anchor of the dehalogenase. This first biochemical and molecular characterization of a chlorophenol reductive dehalogenase has revealed structural resemblance with haloalkene reductive dehalogenases.

AbstractGravity and aeromagnetic data from Britain, Belgium and the southern North Sea have been compiled to provide coverage of the greater part of the Anglo-Brabant Massif. Colour pseudo-relief maps of the gravity and magnetic fields highlight important anomalies and trends which provide new information on the structure of the massif and its margins. Within the massif, prominent SSE-trending geophysical lineaments define the margins of distinctive blocks within the upper crust. These are cross-cut on the northeastern margin of the massif by prominent ESE- and SE-trending magnetic and gravity lineaments. The possible history and origin of the more prominent geophysical anomalies and lineaments are considered. Integrated modelling of the potential field data has been carried out along the BIRPS MOBIL-7 seismic reflection line to provide an interpretation of crustal structure across the northeast margin of the massif constrained by all three datasets. The principal features of the model are a non-reflective, non-magnetic upper crust, interpreted as the Caledonian fold?thrust belt, overlying a heterogeneous middle?lower crust with laterally varying reflectivity, magnetization and density. ESE-trending magnetic anomalies along the northeast edge of the massif are explained in terms of an irregular mid-crustal magnetic layer with a susceptibility comparable to that of the Tubize Group in the Brabant Massif. The top of this body is coincident with prominent dipping mid-crustal reflectors observed on the seismic reflection profile and its overall geometry is compatible with mid-crustal imbrication inferred from the seismic data.

AbstractDigital processing and image-based display techniques have been used to generate contour and shaded-relief maps of Belgian aeromagnetic data at a scale of 1:300000 for the whole of Belgium. These highlight the important anomalies and structural trends, particularly over the Brabant Massif. North and vertically illuminated shaded-relief plots, enhanced structural belts trending west?east to northwest?southeast in the Brabant Massif and west?east to southwest?northeast in the core of the Ardennes. The principal magnetic lineaments have been identified from the shaded-relief plots and tentatively correlated to basement structures. Most short lineaments are correlated with individual folds while the more extensive lineaments are correlated with large scale fault structures. Magnetic highs within the Brabant Massif are attributed to folded sediments of the Tubize Group. The magnetic basement in the east of Belgium is sinistrally displaced to the north by an inferred deep NNW?SSE crustal fracture. The Bouguer anomaly map of Belgium identifies the Ardennes as a negative area, and the Brabant Massif as a positive area, with the exception of a WNW?trending gravity low in its western part. The southern margin of the Brabant Massif is defined by a steep gravity gradient coincident with the Faille Bordiere (Border Fault). Trial modelling of the gravity and magnetic data, carried out along profiles across the Brabant and Stavelot massifs, has identified probable acid igneous intrusions in the western part of the Brabant Massif, and a deep magnetic lower density body underlying the whole Ardennes region, which is thought to be a distinctive Precambrian crustal block.

AbstractA new geological map, mostly subcrop, of the Brabant Massif is presented, based on a revised lithostratigraphy of the outcrop area and on recently acquired palaeontological and lithological data from boreholes in the concealed area. New interpretations of magnetic and gravity data are used to extend the lithostratigraphical units into areas with few or no boreholes. A structural model of the western, northern and southern parts of the Brabant Massif is presented. The main anticlinal axis plunges towards the west-northwest, its core comprising Upper Precambrian (?) to Lower Cambrian terrigenous rocks which outcrop in the southeast. To the north of the main axis, younger rocks appear in regular succession, but to the southwest this picture is complicated by the occurrence of a second anticlinal structure and by a subparallel magmatic arc.

Microorganisms in the environment can often adapt to use xenobiotic chemicals as novel growth and energy substrates. Specialized enzyme systems and metabolic pathways for the degradation of man-made compounds such as chlorobiphenyls and chlorobenzenes have been found in microorganisms isolated from geographically separated areas of the world. The genetic characterization of an increasing number of aerobic pathways for degradation of (substituted) aromatic compounds in different bacteria has made it possible to compare the similarities in genetic organization and in sequence which exist between genes and proteins of these specialized catabolic routes and more common pathways. These data suggest that discrete modules containing clusters of genes have been combined in different ways in the various catabolic pathways. Sequence information further suggests divergence of catabolic genes coding for specialized enzymes in the degradation of xenobiotic chemicals. An important question will be to find whether these specialized enzymes evolved from more common isozymes only after the introduction of xenobiotic chemicals into the environment. Evidence is presented that a range of genetic mechanisms, such as gene transfer, mutational drift, and genetic recombination and transposition, can accelerate the evolution of catabolic pathways in bacteria. However, there is virtually no information concerning the rates at which these mechanisms are operating in bacteria living in nature and the response of such rates to the presence of potential (xenobiotic) substrates. Quantitative data on the genetic processes in the natural environment and on the effect of environmental parameters on the rate of evolution are needed.

Carbon monoxide dehydrogenase (Cdh) has been anaerobically purified from Methanosarcina frisia Gö1. The enzyme is a Ni2+-, Fe2+-, and S2--containing alpha2beta2 heterotetramer of 214 kDa with a pI of 5.2 and subunits of 94 and 19 kDa. It has a Vmax of 0.3 mmol of CO min-1 mg-1 and Km values for CO and methyl viologen of approximately 0.9 mM and 0.12 mM, respectively. EPR spectroscopy on the reduced enzyme showed two overlapping signals: one indicative for 2 (4Fe-4S)+ clusters and a second signal that is atypical for standard Fe/S clusters. The latter was, together with high-spin EPR signals of the oxidized enzyme tentatively assigned to an Fe/S cluster of high nuclearity.

Abstract: Many hyperthermophilic microorganisms show heterotrophic growth on a variety of carbohydrates. There has been considerable fundamental and applied interest in the utilization of glucose and its α- and β-polymers by hyperthermophiles. While glycolysis by Bacteria at high temperatures shows conventional characteristics, it has been found that glucose catabolism by hyperthermophilic Archaea differs from the canonical glycolytic pathways, involves novel enzymes, and shows a unique control. This review addresses these aspects with specific attention to Pyrococcus furiosus, which is one of the best studied hyperthermophilic Archaea, has the capacity to grow on a variety of sugars including the marine β-(1,3)-linked glucose polymer laminarin, and has been found to contain three novel glycolytic enzymes, two ADP-dependent kinases, and a ferredoxin-dependent glyceraldehyde-3-phosphate oxidoreductase.

The plasmid-encoded citrate determinant of the Lactococcus lactis subsp. lactis var. diacetylactis NCDO176 was cloned and functionally expressed in a Cit- Escherichia coli K-12 strain. From deletion derivative analysis, a 3.4-kilobase region was identified which encodes the ability to transport citrate. Analysis of proteins encoded by the cloned fragment in a T7 expression system revealed a 32,000-dalton protein band, which correlated with the ability of cells to transport citrate. Energy-dependent [1,5-14C]citrate transport was found with membrane vesicles prepared from E. coli cells harboring the citrate permease-expressing plasmid. The gene encoding citrate transport activity, citP, was located on the cloned fragment by introducing a site-specific mutation that abolished citrate transport and resulted in a truncated form of the 32,000-dalton expression product. The nucleotide sequence for a 2.2-kilobase fragment that includes the citP gene contained an open reading frame of 1,325 base pairs coding for a very hydrophobic protein of 442 amino acids, which shows no sequence homology with known citrate carriers.

The thermostable β-glucosidase fromPyrococcus furiosushas been shown to produce tri-and tetrasaccharides with lactose as a substrate (0.73–1.44 mol/kg) at elevated temperatures (75–95°C). The enzyme has a broad pH optimum (5.0–7.0), and was inhibited more by glucose than by galactose. An increase in the initial lactose concentrations led to a proportional increase of the ratio between the initial oligosaccharide production rate and the initial lactose conversion rate. Because of inhibition by Maillard components, the lowest temperature studied (75°C) is the best for oligosaccharide synthesis. The oligosaccharide yield obtained with this thermostable enzyme is independent of the initial lactose concentration and temperature and a factor 1.4 higher than reported for mesophilic enzymes.

The fermentative conversion of glucose in anaerobic hyperthermophilic Archaea is a variant of the classical Embden-Meyerhof pathway found in Bacteria and Eukarya. A major difference of the archaeal glycolytic pathway concerns the conversion of glyceraldehyde-3-phosphate. In the hyperthermophilic archaeon Pyrococcus furiosus, this reaction is catalyzed by an unique enzyme, glyceraldehyde-3-phosphate ferredoxin oxidoreductase (GAPOR). Here, we report the isolation, characterization, and transcriptional analysis of the GAPOR-encoding gene. GAPOR is related to a family of ferredoxin-dependent tungsten enzymes in (hyper)thermophilic Archaea and, in addition, to a hypothetical protein in Escherichia coli. Electron paramagnetic resonance analysis of the purified P. furiosus GAPOR protein confirms the anticipated involvement of tungsten in catalysis. During glycolysis in P. furiosus, GAPOR gene expression is induced, whereas the activity of glyceraldehyde-3-phosphate dehydrogenase is repressed. It is discussed that this unprecedented unidirectional reaction couple in the pyrococcal glycolysis and gluconeogenesis gives rise to a novel site of glycolytic regulation that might be widespread among Archaea.

We report here the first molecular characterization of an endo-beta-1,3-glucanase from an archaeon. Pyrococcus furiosus is a hyperthermophilic archaeon that is capable of saccharolytic growth. The isolated lamA gene encodes an extracellular enzyme that shares homology with both endo-beta-1,3- and endo-beta-1,3-1,4-glucanases of the glycosyl hydrolase family 16. After deletion of the N-terminal leader sequence, a lamA fragment encoding an active endo-beta-1,3-glucanase was overexpressed in Escherichia coli using the T7-expression system. The purified P. furiosus endoglucanase has highest hydrolytic activity on the beta-1,3-glucose polymer laminarin and has some hydrolytic activity on the beta-1,3-1,4 glucose polymers lichenan and barley beta-glucan. The enzyme is the most thermostable endo-beta-1,3-glucanase described up to now; it has optimal activity at 100-105 degrees C. In the predicted active site of glycosyl hydrolases of family 16 that show predominantly endo-beta-1,3-glucanase activity, an additional methionine residue is present. Deletion of this methionine did not change the substrate specificity of the endoglucanase, but it did cause a severe reduction in its catalytic activity, suggesting a structural role of this residue in constituting the active site. High performance liquid chromatography analysis showed in vitro hydrolysis of laminarin by the endo-beta-1,3-glucanase proceeds more efficiently in combination with an exo-beta-glycosidase from P. furiosus (CelB). This most probably reflects the physiological role of these enzymes: cooperation during growth of P. furiosus on beta-glucans.

Biosynthesis of the lantibiotic peptide nisin by Lactococcus lactis NIZO R5 relies on the presence of the conjugative transposon Tn5276 in the chromosome. A 12-kb DNA fragment of Tn5276 including the nisA gene and about 10 kb of downstream DNA was cloned in L. lactis, resulting in the production of an extracellular nisin precursor peptide. This peptide reacted with antibodies against either nisin A or the synthetic leader peptide, suggesting that it consisted of a fully modified nisin with the nisin leader sequence still attached to it. This structure was confirmed by N-terminal sequencing and 1H-nuclear magnetic resonance analysis of the purified peptide. Deletion studies showed that the nisR gene is essential for the production of this intermediate. The deduced amino acid sequence of the nisR gene product indicated that the protein belongs to the family of two-component regulators. The deduced amino acid sequence of NisP, the putative product of the gene upstream of nisR, showed an N-terminal signal sequence, a catalytic domain with a high degree of similarity to those of subtilisin-like serine proteases, and a putative C-terminal membrane anchor. Cell extracts of Escherichia coli overexpressing nisP were able to cleave the nisin precursor peptide, producing active, mature nisin. A similar activation was obtained with whole cells but not with membrane-free extracts of L. lactis strains carrying Tn5276 in which the nisA gene had been inactivated. The results indicate that the penultimate step in nisin biosynthesis is secretion of precursor nisin without cleavage of the leader peptide, whereas the last step is the cleavage of the leader peptide sequence from the fully maturated nisin peptide.

The dnaJ gene of Lactococcus lactis was isolated from a genomic library of L. lactis NIZO R5 and cloned into pUC19. Nucleotide sequencing revealed an open reading frame of 1,137 bp in length, encoding a protein of 379 amino acids. The deduced amino acid sequence showed homology to the DnaJ proteins of Escherichia coli, Mycobacterium tuberculosis, Bacillus subtilis, and Clostridium acetobutylicum. The level of the dnaJ monocistronic mRNA increased approximately threefold after heat shock. The transcription initiation site of the dnaJ gene was determined and appeared to be preceded by a typical gram-positive vegetative promoter sequence (TTGCCA-17 bp-TAAAAT). Upstream of the promoter region, an inverted repeat is located that is identical to those detected upstream of heat shock genes of other gram-positive organisms. A transcriptional fusion between the dnaJ expression signals and a usp45-amyS secretion cassette caused a significant increase in alpha-amylase activity after heat shock induction. Deletion mutagenesis showed that the inverted repeat is involved in heat shock regulation of the dnaJ gene. The conservation of this palindromic sequence in gram-positive heat shock genes suggests a common regulatory pathway distinct from the system used in gram-negative bacteria.

We determined the location, activity, and regulation of the promoter of the Lactococcus lactis 8-kb lactose operon (lacABCDFEGX), which encodes the enzymes of the lactose phosphotransferase system and the tagatose 6-phosphate pathway. The lac promoter sequence corresponds closely to the consensus promoter described for gram-positive bacteria and is located in a back-to-back configuration with the promoter of the divergently transcribed lacR gene, which encodes the LacR repressor. The transcription start sites used under induced (lactose) and noninduced (glucose) conditions were determined. The minimal promoter region that could be isolated on a single restriction fragment included sequences ranging from -75 to +42. The effect of the presence of flanking sequences and the lacR gene on promoter activity and regulation was studied in Escherichia coli and L. lactis strains by using transcriptional fusions with promoterless chloramphenicol acetyltransferase reporter genes. The results showed that transcriptional regulation of the lac operon is mediated by the interaction between the LacR repressor, the lac promoter, and sequences in the noncoding region between the lacR and lacA genes. Sequences flanking the minimal promoter region appeared to enhance lac promoter activity much more in L. lactis (5- to 38-fold) than in E. coli (1.3- to 5-fold).

A 16-kb BamHI fragment of the lactose plasmid pNZ63 from Leuconostoc lactis NZ6009 was cloned in Escherichia coli MC1061 by using pACYC184 and was found to express a functional beta-galactosidase. Deletion and complementation analysis showed that the coding region for beta-galactosidase was located on a 5.8-kb SalI-BamHI fragment. Nucleotide sequence analysis demonstrated that this fragment contained two partially overlapping genes, lacL (1,878 bp) and lacM (963 bp), that could encode proteins with calculated sizes of 72,113 and 35,389 Da, respectively. The L. lactis beta-galactosidase was overproduced in E. coli by using a lambda pL expression system. Two new proteins with M(r)s of 75,000 and 36,000 appeared upon induction of PL. The N-terminal sequences of these proteins corresponded to those deduced from the lacL and lacM gene sequences. Mutation and deletion analysis showed that lacL expression is essential for LacM production and that both the lacL and lacM genes are required for the production of a functional beta-galactosidase in E. coli. The deduced amino acid sequences of the LacL and LacM proteins showed considerable identity with the sequences of the N- and C-terminal parts, respectively, of beta-galactosidases from other lactic acid bacteria or E. coli. DNA and protein sequence alignments suggest that the L. lactis lacL and lacM genes have been generated by an internal deletion in an ancestral beta-galactosidase gene.

The plasmid-encoded lactose genes of the Lactococcus lactis phosphotransferase system encoding Enzyme IIIlac (lacF) and Enzyme IIlac (lacE) have been identified and cloned in Escherichia coli and L. lactis. Nucleotide sequence and transcription analysis showed that these genes are organized into a lactose-inducible operon with the gene order lacF-lacE-lacG-lacX, the latter two genes encoding phospho-beta-galactosidase and a 34-kDa protein with an unknown function, respectively. The lac-operon is immediately followed by an IS element that is homologous to ISS1. Enzyme IIIlac was purified from L. lactis and determination of its NH2-terminal sequence demonstrated that the lacF gene starts with a TTG codon and encodes a 105 amino acid protein (Mr = 11416). Cross-linking studies with the purified enzyme showed that Enzyme IIIlac is active as a trimer. A mutant lacF gene was identified in strain YP2-5 and appeared to encode Enzyme IIIlac containing the missense mutation G18E. The lacF gene could be expressed under control of vector-located promoter sequences resulting in overproduction of Enzyme IIIlac in E. coli and complementation of the L. lactis lacF mutant YP2-5. The deduced amino acid sequence of Enzyme IIlac consists of 586 amino acids (Mr = 61562) and shows the characteristics of a hydrophobic, integral membrane protein. The deduced primary structures of the L. lactis Enzyme IIIlac and Enzyme IIlac are homologous to those of Staphylococcus aureus (72 and 71% identity, respectively) and Lactobacillus casei (48 and 47% identity, respectively). In contrast, the organization of the lactose genes differs significantly between those Gram-positive bacteria. Heterogramic homology in specific domains was observed between the derived amino acid sequences of the lactose-specific enzymes and that of E. coli Enzyme IIIcel and Enzyme IIcel, which suggest a common function in the transport and phosphorylation of these structurally related beta-glucosides.

The catalytic domain of the cell-envelope proteinase from Lactococcus lactis SK11 has various inserts, situated in external loops of the catalytic domain, compared with the related subtilisins. Protein engineering was employed to analyse the necessity and function of one of these extra loops (residues 205-219), that is predicted to be located in close proximity to the substrate-binding region and is susceptible to autoproteolysis. We constructed a deletion mutant which lacks 14 residues of this surface loop and subsequently introduced various insertion cassettes coding either for the original loop with three mutations (E205S/E218T/M219S: triple-mutant proteinase) or for neutral spacers (1, 4, 7 and 16 serine residues). Engineered proteinases were analysed for activity, (auto)processing, and cleavage specificity. The presence of residues 205-219 is shown to be essential for proteolytic activity, as only triple-mutant proteinase retained activity towards casein substrates. The triple-mutant proteinase was found to be defective in C-terminal autoprocessing, and subsequent release from the lactococcal cell envelope in a calcium-free medium, indicative of either an altered proteolytic specificity or altered accessibility of the processing site. The specificity change appears to be subtle, as only small differences were found between wild-type and triple-mutant proteinase in the breakdown of casein substrates.

Insertions, replacement mutations, and deletions were introduced via single or double crossover recombination into the lacE (enzyme IIlac) and lacG (phospho-beta-galactosidase) genes of the Lactococcus lactis chromosomal lacABCDFEGX operon. LacG production was abolished in strains missing the lacG gene or carrying multicopy insertions in the lacE gene that affected expression of the lacG gene. However, these LacG-deficient strains could still ferment lactose slowly and were found to contain an enzymatic activity that hydrolyzed the chromogenic substrate o-nitrophenyl-beta-D-galactopyranoside phosphate. Induction of this phospho-beta-glycohydrolase activity coincided with the appearance of a new 55-kDa protein cross-reacting with anti-LacG antibodies that had a size similar to that of LacG but a higher isoelectric point (pI 5.2) and was not found in wild-type cells during growth on lactose. Since the phospho-beta-glycohydrolase activity and this protein with a pI of 5.2 were highly induced in both mutant and wild-type cells during growth on cellobiose that is likely to be transported via a phosphoenolpyruvate-dependent phosphotransferase system, we propose that this induced activity is a phospho-beta-glucosidase that also hydrolyzes lactose-6-phosphate.

A restriction-modification system, designated MthTI, was localized on plasmid pFV1 from the thermophilic archaeon Methanobacterium thermoformicicum THF. The MthTI system is a new member of the family of GGCC-recognizing restriction-modification systems. Functional expression of the archaeal MthTI genes was obtained in Escherichia coli. The mthTIR and mthTIM genes are 843 and 990 bp in size and code for proteins of 281 (32,102 Da) and 330 (37,360 Da) amino acids, respectively. The deduced amino acid sequence of M.MthTI showed high similarity with that of the isospecific methyltransferases M.NgoPII and M.HaeIII. In addition, extensive sequence similarity on the amino acid level was observed for the endonucleases R.MthTI and R.NgoPII. Moreover, the endonuclease and methyltransferase genes of the thermophilic MthTI system and those of the Neisseria gonorrhoeae NgoPII system show identical organizations and high (54.5%) nucleotide identity. This finding suggests horizontal transfer of restriction-modification systems between members of the domains Bacteria and Archaea.

A novel, chromosomally located conjugative transposon in Lactococcus lactis, Tn5276, was identified and characterized. It encodes the production of and immunity to nisin, a lanthionine-containing peptide with antimicrobial activity, and the capacity to utilize sucrose via a phosphotransferase system. Conjugal transfer of Tn5276 was demonstrated from L. lactis NIZO R5 to different L. lactis strains and a recombination-deficient mutant. The integration of Tn5276 into the plasmid-free strain MG1614 was analyzed by using probes based on the gene for the nisin precursor (nisA) and the gene for sucrose-6-phosphate hydrolase (sacA). The transposon inserted at various locations in the MG1614 chromosome and showed a preference for orientation-specific insertion into a single target site (designated site 1). By using restriction mapping in combination with field inversion gel electrophoresis and DNA cloning of various parts of the element including its left and right ends, a physical map of the 70-kb Tn5276 was constructed, and the nisA and sacA genes were located. The nucleotide sequences of Tn5276 junctions in donor strain NIZO R5 and in site 1 of an MG1614-derived transconjugant were determined and compared with that of site 1 in recipient strain MG1614. The results show that the A + T-rich ends of Tn5276 are flanked by a direct hexanucleotide repeat in both the donor and the transconjugant but that the element does not contain a clear inverted repeat.

Plasmid pP51 of Pseudomonas sp. strain P51 contains two gene clusters encoding the degradation of chlorinated benzenes, tcbAB and tcbCDEF. A regulatory gene, tcbR, was located upstream and divergently transcribed from the chlorocatechol oxidative gene cluster tcbCDEF. The tcbR gene was characterized by DNA sequencing and expression studies with Escherichia coli and pET8c and appeared to encode a 32-kDa protein. The activity of the tcbR gene product was analyzed in Pseudomonas putida KT2442, in which it appeared to function as a positive regulator of tcbC expression. Protein extracts of both E. coli overproducing TcbR and Pseudomonas sp. strain P51 showed specific DNA binding to the 150-bp region that is located between the tcbR and tcbC genes. Primer extension mapping demonstrated that the transcription start sites of tcbR and tcbC are located in this region and that the divergent promoter sequences of both genes overlap. Amino acid sequence comparisons indicated that TcbR is a member of the LysR family of transcriptional activator proteins and shares a high degree of homology with other activator proteins involved in regulating the metabolism of aromatic compounds.

The repressor gene (lacR) of the lactose phosphotransferase system of Lactococcus lactis subsp. lactis strain MG1820 has been cloned and characterized. Transcription of lacR, into a 1.2-kilobase monocistronic messenger, is repressed approximately 5-fold during growth on lactose. Nucleotide sequence analysis of the lacR gene showed the presence of an open reading frame of 861 base pairs. The deduced amino acid sequence of LacR is homologous to three Escherichia coli regulatory proteins (DeoR, FucR, and GutR) and includes a N-terminal domain (helix-turn-helix) involved in DNA binding and a C-terminal domain that may be responsible for inducer binding. The in vivo function of LacR has been determined by introducing multiple copies of lacR into L. lactis, under control of its own or the unrelated prtP promoter. Growth rates and lactose phosphotransferase system enzyme activities were measured during growth on lactose and glucose. The presence of lacR on a multicopy plasmid resulted in the decrease of lactose phosphotransferase system activity, whereas only on lactose a decrease (25%) of growth rate was observed. No significant difference in growth rate was observed on glucose, indicating that LacR specifically represses the lactose genes of L. lactis.

Analysis of one of the regions of catabolic plasmid pP51 which encode chlorobenzene metabolism of Pseudomonas sp. strain P51 revealed that the tcbA and tcbB genes for chlorobenzene dioxygenase and dehydrogenase are located on a transposable element, Tn5280. Tn5280 showed the features of a composite bacterial transposon with iso-insertion elements (IS1066 and IS1067) at each end of the transposon oriented in an inverted position. When a 12-kb HindIII fragment of pP51 containing Tn5280 was cloned in the suicide donor plasmid pSUP202, marked with a kanamycin resistance gene, and introduced into Pseudomonas putida donor plasmid pSUP202, marked with a kanamycin resistance gene, and introduced into Pseudomonas putida KT2442, Tn5280 was found to transpose into the genome at random and in single copy. The insertion elements IS1066 and IS1067 differed in a single base apir located in the inner inverted repeat and were found to be highly homologous to a class of repetitive elements of Bradyrhizobium japonicum and distantly related to IS630 of Shigella sonnei. The presence of the catabolic genes tcbA and tcbB on Tn5280 suggests a mechanism by which gene clusters can be mobilized as gene cassettes and joined with others to form novel catabolic pathways.

In the acetoclastic methanogen Methanothrix soehngenii, acetate is activated to acetyl coenzyme A by acetyl coenzyme A synthetase (Acs). The acs gene, coding for the single Acs subunit, was isolated from a genomic library of M. soehngenii DNA in Escherichia coli by using antiserum raised against the purified Acs. After introduction in E. coli, the acs gene was expressed, resulting in the production of an immunoreactive protein of 68 kDa, which is approximately 5 kDa smaller than the known size of purified Acs. In spite of this difference in size, the Acs enzymes are produced in similar quantities in E. coli and M. soehngenii and show comparable specific activities. Upstream from the acs gene, consensus archaeal expression signals were identified. Immediately downstream from the acs gene there was a putative transcriptional stop signal. The amino acid sequence deduced from the nucleotide sequence of the acs gene showed homology with those of functionally related proteins, i.e., proteins involved in the binding of coenzyme A, ATP, or both.

Pseudomonas sp. strain P51 contains two gene clusters located on catabolic plasmid pP51 that encode the degradation of chlorinated benzenes. The nucleotide sequence of a 5,499-bp region containing the chlorocatechol-oxidative gene cluster tcbCDEF was determined. The sequence contained five large open reading frames, which were all colinear. The functionality of these open reading frames was studied with various Escherichia coli expression systems and by analysis of enzyme activities. The first gene, tcbC, encodes a 27.5-kDa protein with chlorocatechol 1,2-dioxygenase activity. The tcbC gene is followed by tcbD, which encodes cycloisomerase II (39.5 kDa); a large open reading frame (ORF3) with an unknown function; tcbE, which encodes hydrolase II (25.8 kDa); and tcbF, which encodes a putative trans-dienelactone isomerase (37.5 kDa). The tcbCDEF gene cluster showed strong DNA homology (between 57.6 and 72.1% identity) and an organization similar to that of other known plasmid-encoded operons for chlorocatechol metabolism, e.g., clcABD of Pseudomonas putida and tfdCDEF of Alcaligenes eutrophus JMP134. The identity between amino acid sequences of functionally related enzymes of the three operons varied between 50.6 and 75.7%, with the tcbCDEF and tfdCDEF pair being the least similar of the three. Measurements of the specific activities of chlorocatechol 1,2-dioxygenases encoded by tcbC, clcA, and tfdC suggested that a specialization among type II enzymes has taken place. TcbC preferentially converts 3,4-dichlorocatechol relative to other chlorinated catechols, whereas TfdC has a higher activity toward 3,5-dichlorocatechol. ClcA takes an intermediate position, with the highest activity level for 3-chlorocatechol and the second-highest level for 3,5-dichlorocatechol.

Pseudomonas sp. strain P51 is able to use 1,2-dichlorobenzene, 1,4-dichlorobenzene, and 1,2,4-trichlorobenzene as sole carbon and energy sources. Two gene clusters involved in the degradation of these compounds were identified on a catabolic plasmid, pP51, with a size of 110 kb by using hybridization. They were further characterized by cloning in Escherichia coli, Pseudomonas putida KT2442, and Alcaligenes eutrophus JMP222. Expression studies in these organisms showed that the upper-pathway genes (tcbA and tcbB) code for the conversion of 1,2-dichlorobenzene and 1,2,4-trichlorobenzene to 3,4-dichlorocatechol and 3,4,6-trichlorocatechol, respectively, by means of a dioxygenase system and a dehydrogenase. The lower-pathway genes have the order tcbC-tcbD-tcbE and encode a catechol 1,2-dioxygenase II, a cycloisomerase II, and a hydrolase II, respectively. The combined action of these enzymes degrades 3,4-dichlorocatechol and 3,4,6-trichlorocatechol to a chloromaleylacetic acid. The release of one chlorine atom from 3,4-dichlorocatechol takes place during lactonization of 2,3-dichloromuconic acid.

The complete nucleotide sequence of a gene located immediately upstream of the Lactococcus lactis subsp. cremoris SK11 prtP gene encoding the cell envelope-attached proteinase was determined. This gene, designated prtM, was found to be transcribed from the same promotor region as was the proteinase gene but in the opposite direction. The prtM gene directed the expression in Escherichia coli of a protein with a size similar to the expected value of 33 kilodaltons, as deduced from the nucleotide sequence data. The derived amino acid sequence of the PrtM protein indicated the presence of a consensus lipoprotein signal sequence at the N terminus, which suggested that PrtM is a lipoprotein. Plasmids containing the prtM gene, the prtP gene, or both were constructed. Expression studies of L. lactis clones containing these plasmids showed that the prtM gene encodes a trans-acting activity involved in the maturation of cell envelope-located and -secreted forms of the SK11 proteinase.

The tagatose 6-phosphate pathway gene cluster (lacABCD) encoding galactose-6-phosphate isomerase, tagatose-6-phosphate kinase, and tagatose-1,6-diphosphate aldolase of Lactococcus lactis subsp. lactis MG1820 has been characterized by cloning, nucleotide sequence analysis, and enzyme assays. Transcription studies showed that the four tagatose 6-phosphate pathway genes are the first genes of the lactose-inducible lactose-phosphotransferase operon consisting of the lacABCDFEGX genes. Using a T7 expression system, it could be shown that the lacA, lacB, lacC, and lacD genes code for proteins with apparent molecular masses of 15, 19, 33, and 36 kDa, respectively. Cell-free extracts of induced and noninduced Escherichia coli cells expressing the lacABCD genes were used to determine the functions of the encoded proteins. Expression of both lacA and lacB was required to obtain galactose-6-phosphate isomerase activity. The lacC gene codes for tagatose-6-phosphate kinase, the deduced amino sequence of which is similar to that of E. coli Pfk-2 phosphofructokinase, and Staphylococcus aureus LacC protein. The tagatose-1,6-diphosphate aldolase is encoded by the lacD gene, and its deduced primary sequence, which is homologous to that of the S. aureus LacD protein, predicts an amino acid composition which is virtually identical to that of the previously purified L. lactis E8 tagatose-1,6-diphosphate aldolase.

The cdhA and cdhB genes that code for the large and the small subunits of carbon monoxide dehydrogenase (CDH), respectively, were isolated from a genomic library of Methanothrix soehngenii DNA in Escherichia coli, using polyclonal antibodies raised against purified CDH. After introduction in E. coli or Desulfovibrio vulgaris, the cdh genes appeared to be expressed irrespective of their orientation, yielding immunoreactive proteins of 79 and 19 kDa, corresponding in size to the known subunits of purified CDH. However, no CDH activity could be detected in these heterologous hosts. The cdh genes are preceded by consensus ribosome-binding sites and are arranged in an operon-like structure, with cdhA preceding cdhB. Upstream from this operon, sequences similar to archaeal promoters were identified. The amino acid sequence, deduced from the primary sequence of cdhA, showed homology with ferredoxins and with acyl-CoA oxidase. This is compatible with the proposed functions of CDH.

The post-translationally modified, antimicrobial peptide nisin is secreted by strains of Lactococcus lactis that contain the chromosomally located nisin biosynthetic gene cluster nisABTCIPRKFEG. When a 4-base pair deletion is introduced into the structural nisA gene (delta nisA), transcription of delta nisA is abolished. Transcription of the delta nisA gene is restored by adding subinhibitory amounts of nisin, nisin mutants, or nisin analogs to the culture medium, but not by the unmodified precursor peptide or by several other antimicrobial peptides. Upon disruption of the nisK gene, which encodes a putative sensor protein that belongs to the class of two-component regulators, transcription of delta nisA was no longer inducible by nisin. Fusion of a nisA promoter fragment to the promoterless reporter gene gusA resulted in expression of gusA in L. lactis NZ9800 (delta nisA) only upon induction with nisin species. The expression level of gusA was directly related to the amount of inducer that was added extracellularly. These results provide insight into a new mechanism of autoregulation through signal transduction in prokaryotes and demonstrate that antimicrobial peptides can exert a second function as signaling molecules.

Transformation-deficient mutants of Bacillus subtilis were selected after replica plating on agar plates containing transforming DNA. Out of 24 mutants tested, 3 showed highly reduced abilities to bind donor DNA; the residual levels of binding were similar to those of noncompetent cells. Transformation and transfection were reduced to nondetectable levels in the mutants. However, transduction with phage SPP1 occurred at normal frequencies. The nuclease activities involved in entry of donor DNA were present in the mutants. Comparison of protein patterns by two-dimensional gel electrophoresis revealed the absence of one major protein in the mutants as compared with the wild-type strain. This protein (molecular weight, approximately 18,000; isoelectric point, 5.0) appeared to be membrane associated. The protein was specific for competent cells, suggesting that it is involved in the binding of donor DNA.

We have determined the complete nucleotide sequence of the gene for the cell envelope-located proteinase of Lactococcus lactisSK11. The gene contains a very AT-rich promoter region followed by the coding sequence of a protein of 1962 amino acids. Comparison of the NH2-terminal amino acid sequence of the mature proteinase and the expected primary translation product of the proteinase gene indicates that the enzyme is probably synthesized as a pre-pro-protein. This is confirmed by expression studies of the proteinase gene in Escherichia coli. The amino acid sequence of the proteinase shows significant homology to a number of serine proteinases of the subtilisin family. Compared with the related proteinase of L. lactisWg2, the proteinase of L. lactis SK11 contains a 60-amino acids duplication and a total of 44-amino acid substitutions, some of which may account for the different cleavage specificity of both enzymes. Furthermore, a region was identified in the Lactococcus proteinase, which shows homology to the membrane-anchoring domains of a number of proteins from other Gram-positive bacteria.